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Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.
Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.
Naturschutz-Info 2/2007
(2007)
Naturschutz-Info 1/2007
(2007)
Anfang Juli 1797 hat Schiller das von ihm für den nächsten Almanach zur Publikation vorgesehene Gedicht "Nadowessische Totenklage" an drei Freunde zur Beurteilung geschickt, an Wilhelm von Humboldt, [...] an Körner in Dresden und an Goethe in Weimar. [...] Als Grund für diese Art von Netzwerkbildung lässt sich ein Orientierungsnotstand bezeichnen. Da Schiller unentwegt poetisches Neuland betreten wollte, war er neben theoretischer Reflexion auf die bestätigend-kritische Absicherung durch das Urteil seiner Freunde angewiesen. Schiller überraschte seine Freunde immer wieder mit neuartigen poetischen Versuchen. Auch dieses Mal im Fall der "Nadowessischen Totenklage" hat Goethe sofort erkannt und anerkannt, dass hiermit der "Kreis der poetischen Gegenstände" erweitert worden sei.